鑑定非典型端粒延長癌症中的TERRA RNA交互作用蛋白及人類肺癌細胞中SARS-CoV-2病毒核酸相互作用蛋白質

Abstract

大部分癌細胞透過活化端粒酶(telomerase)來延長端粒，但有10~15%的癌細胞依靠一種稱為非典型端粒延長(Alternative Lengthening of Telomeres, 簡稱ALT)的機制來延長端粒。目前已知ALT會透過同源重組(homologous recombination, HR)及斷裂誘導DNA合成的機制來促進端粒延長，並且其中有TERRA (telomeric repeat-containing RNA) RNA的參與。TERRA是一種在ALT癌細胞裡高度表現的長鏈非編碼RNA (long non-coding RNA, lncRNA)，能與端粒DNA形成RNA: DNA相連的複合結構（此結構中包含R-loop）。然而TERRA如何參與ALT的機制仍不明確。為了研究TERRA所扮演的角色，我們利用iDRiP-MS (identification of direct RNA-interacting proteins mass spectrometry，RNA 相互作用蛋白質譜鑑定)方法在ALT癌細胞體內鑑定TERRA相互作用蛋白質，這是一種通過紫外線交聯（UV-crosslinking）來捕獲RNA相互作用蛋白的方法。結果顯示TERRA與許多DNA損傷反應、DNA修復、複製叉拆解和DNA複製相關蛋白具有交互作用。我們接著透過過量表達RNase H1，發現消除TERRA R-loop會導致ALT活性受到抑制以及端粒聚集現象（telomere clustering）的減少。而剔除RNase H1則會促進ALT活性並使端粒聚集現象增加。我們並發現剔除RNase H1所導致的TERRA R-loop增加，會促進TERRA 相互作用蛋白（BLM、XPF 和 RPA70）向 ALT 端粒募集，這意味著TERRA R-loop會透過增加端粒配對和向端粒招募 DNA 損傷反應蛋白來促進同源重組。我們的研究表明TERRA與DNA損傷反應蛋白相互作用，並調節ALT癌細胞端粒的延長。 新型冠狀病毒（SARS-CoV-2）在短時間內引發了嚴重大流行，導致全球經濟、健康和社會的問題。由於其快速傳播，迄今已積累大量的突變株。病毒變異可能導致更具傳染性以及能逃避疫苗的病毒株；因此，研究SARS-CoV-2進化機制以及宿主的環境是如何參與其中至關重要。為了研究SARS-CoV-2及其宿主的交互作用，我們通過iDRiP-MS系統化地捕獲人類肺癌細胞（Calu3細胞）中與SARS-CoV-2進行交互作用的蛋白質，我們找到了涉及多種途徑的蛋白質群，包括轉譯作用（例如 EIF4B、EIF4H、LARP1、CSDE1）、RNA 編輯（例如 A1CF、RBM47、APOBEC3F）、應激顆粒組成（例如 TIAL1、G3BP1、IGF2BP3) 和免疫反應相關蛋白。透過基因剔除實驗，我們發現其中兩個交互作用蛋白APOBEC1和A1CF的缺失會使Calu3細胞中的SARS-CoV-2 RNA減少，顯示其可能促進病毒複製，而APOBEC3F和RBM47則呈現相反結果，顯示其可能抑制病毒複製。我們亦發現，APOBEC3F在人類肺癌細胞中的表現量會受到感染SARS-CoV-2的影響而上調。我們的研究結果為進一步研究 SARS-CoV-2 病毒適應性和復制機制提供了有價值的信息。

Most cancers activate telomerase to elongate their telomeres; however, a fraction of cancer cells depends on a mechanism called alternative lengthening of telomere (ALT) to compensate for telomere loss to reach immortality. It is commonly accepted that this mechanism is achieved by raising the homologous recombination and break-induced DNA synthesis among telomeres, with the participation of telomeric repeat-containing RNA (TERRA). TERRA is a long non-coding RNA (lncRNA) highly expressed in ALT cells and can form RNA: DNA hybrids (including R-loop structure) at ALT telomeres. However, how TERRA involves in human ALT tumors remains unclear. To investigate the role of TERRA in the ALT mechanism, we identified TERRA interactomes in vivo by performing iDRiP (identification of direct RNA-interacting proteins), which captures RNA-interacting proteins by ultra-light-crosslinking of RNAs and proteins in human ALT cells. We found a subset of TERRA interactomes relative to DNA damage response, DNA repair, fork processing, and DNA replication. We then found that eliminating TERRA R-loops by overexpression of RNase H1 led to the inhibition of ALT activity and the reduction of telomere clustering events, while depletion of RNase H1 increases ALT activity and telomere clustering events. Moreover, the upregulation of telomeric R-loops caused by the depletion of RNase H1 promotes the recruitment of TERRA interacting proteins—BLM, XPF, and RPA70 to ALT telomeres, implying that TERRA R-loops promote homologous recombination via increasing telomere pairing. In sum, our studies suggest that TERRA associates with DNA damage-responsive proteins and regulates telomere lengthening in ALT cancer cells. The SARS-CoV-2 virus has caused a severe pandemic shortly, leading to serious economic, health, and social problems worldwide. It has accumulated numerous mutant strains due to its rapid spreading. Virus variability can cause more infectious and vaccine-evading virus strains; hence, it is crucial to investigate the mechanism of SARS-CoV-2 evolution and how the host machinery is involved. To study the interplay between SARS-CoV-2 and its host, we systematically captured SARS-CoV-2 interacting proteins in vivo by iDRiP-MS in Calu3 cells, a human lung cancer cell line, and revealed SARS-CoV2 interacting proteins that involve in a large variety of pathways including translations (e.g. EIF4B, EIF4H, LARP1, CSDE1), RNA editing (e.g. A1CF, RBM47, APOBEC3F), stress granule composition (e.g. TIAL1, G3BP1, IGF2BP3), and immune response. Silencing SARS-CoV-2 interacting proteins APOBEC1 and A1CF decreased the amount of SARS-CoV-2 in Calu3 cells, suggesting they might promote viral replication. In contrast, depletion of APOBEC3F and RBM47 showed the opposite result, suggesting that they might inhibit viral replication. Furthermore, we found that the expression of APOBEC3F in human lung cancer cells is upregulated upon infection, suggesting its function in SARS-CoV-2 RNA viral progeny inhibition. Our study provides valuable information for further investigations into SARS-CoV-2 viral fitness and replication mechanism.